Chi hotspot control of RecBCD helicase-nuclease by long-range intramolecular signaling

Abstract

Life-saving repair of broken DNA by homologous recombination requires coordinated enzymatic reactions to prepare it for interaction with intact DNA. The multiple activities of enterobacterial RecBCD helicase-nuclease are coordinated by Chi recombination hotspots (5' GCTGGTGG 3') recognized during DNA unwinding. Our previous studies led to a signal transduction model in which the Chi-bound RecC subunit signals RecD, a fast helicase, to stop; RecD then signals RecB, a slower helicase, to swing its nuclease domain into position to cut DNA at Chi and load RecA protein onto the newly generated 3'-ended single-stranded DNA. Here, we report a set of mutants blocked at each step in this pathway. The mutants have helicase and degradative nuclease activities, but most have lost or reduced coordination by Chi and have reduced recombination and DNA repair proficiency. The mutant alterations are widely scattered throughout the three subunits of RecBCD; each alters the intimate contact between two subunits in the crystal or cryoEM structures, supporting the signal transduction model. Similar large conformational changes in other vital enzymes, such as DNA and RNA polymerases, may make RecBCD a paradigm for elucidating their control by the nucleic acid sites they recognize. Author summaryRepair of broken DNA is essential for life. Faithful repair occurs by homologous genetic recombination, which also generates new gene allele combinations important for evolution. In bacteria the RecBCD helicase-nuclease enzyme is essential for most DNA break repair and is regulated by special DNA sites (Chi recombination hotspots). As it unwinds DNA, RecBCD's activities are dramatically altered at Chi to prepare the broken DNA for interaction with intact DNA to finish repair. Here, we describe RecBCD mutants that respond weakly, or not at all, to Chi; the alterations are scattered widely throughout the enzyme, supporting our previous model of large conformational changes of RecBCD at Chi. Similar methods may help understand how other large enzyme complexes are regulated.

Small-molecule sensitization of RecBCD helicase–nuclease to a Chi hotspot-activated state

“…The mechanism for sensitization of RecB to cut the DNA when RecD stops is less obvious. Recent analysis of mutations in each subunit that reduce or block Chi hotspot activity shows that amino acids widely scattered throughout the large RecBCD complex are important for Chi to signal DNA cutting ( 19 ). Three points of contact (RecC–RecD, RecD–RecB and RecB–RecC) appear to act sequentially to transmit the Chi signal from the RecC tunnel to the RecB nuclease domain (Figure 1B – D ).…”

“…Extensive mutational analyses of the RecC tunnel and of the RecB tether support this model ( 17 , 18 , 16 ). Mutants with altered contacts between RecC–RecD, RecD–RecB and RecB–RecC indicate that each of these contacts is important for Chi hotspot activity ( 19 ).…”